The central theme of this project is a study of lipid-protein interactions in biological membranes with the eventual aim of understanding membranes of normal and malignant tissues. The strategy is to introduce spin label analogues of specific naturally occurring membrane phospholipids into preparations containing a single kind of functional protein, and to study lipid mobility and distribution by electron spin resonance. In previous work we introduce new classes of lipid spin labels, developed the necessary computer data analytical techniques, and established (in the case of the integral membrane protein cytochrome oxidase) that a constant amount of lipid is immobilized on the hydrophobic surface of the protein complex in equilibrium with the adjacent bilayer. To exploit the advances made so far, we propose to examine the lipid-protein associations in membranes reconstituted from purified membrane proteins and defined phospholipids. Specifically, our objectives are to study the three integral membrane proteins Na/K-ATPase, Ca-ATPase and cytochrome oxidase reconstituted in defined lipids. By correlating ESR data, NMR data, activity measurements, electron microscopy and lipid analyses, it will be possible to characterize the specific interactions of these membrane proteins with phospholipids differing in head group and fatty acid chain composition.